My voyage in the enchanted world of sleep.

In this paper, I describe my 45-year career in sleep research. I started my undergraduate studies at Tel Aviv University, where I was first introduced to the enchanted world of sleep, continued to my graduate studies with Wilse B. Webb at the University of Florida, and then to post-doctoral training with Dan Kripke at the University of California at San Diego. Then, I describe the evolution of my academic career at the Technion-Israel Institute of Technology, where I started in 1975 as an Assistant Professor and retired in 2019 as the President of the Institute. I describe the areas of research that I pursued and how the research developed, emphasizing unexpected results that guided me and my lab team in new directions. This includes my early studies on ultradian rhythms, inspired by Nathaniel Kleitman's Basic Rest Activity Cyle hypothesis, utilizing the ultrashort sleep-wake paradigm to chart the 24-hour sleep propensity function, and how these studies led us to explore the role of melatonin in sleep regulation. I also explain why we directed our attention to sleep apnea, and how clinical observations led to the provocative hypothesis that sleep apnea-typically seen as a disorder-may also play a protective role. Under the leadership of my research partner and wife, Lena, we confirmed this hypothesis. Also in this article, I describe my enthusiasm for the history of our field and, as derived from my experience as a Dean of Medicine and President of a university, I share my philosophy about the role of members of academia in society. I emphasize that none of my achievements could have been accomplished without the hard work and motivation of my students and research partners, who shared my enthusiasm and passion for the enchanted world of sleep. This paper is part of the Living Legends in Sleep Research series, which is sponsored by Idorsia Pharmaceuticals and Jazz Pharmaceuticals.

Near-total absence of REM sleep co-occurring with normal cognition: an update of the 1984 paper.

BACKGROUND: REM sleep (REMS) is considered vital for supporting well-being and normal cognition. However, it remains unclear if and how decreases in REMS impair cognitive abilities. Rare case studies of patients with REMS abolishment due to pontine lesions remain sporadic, and formal evaluation of cognitive status is lacking. In 1984, Lavie and colleagues described the case of Y.C. - a man with a pontine lesion and near-total absence of REMS who led a normal life. Here, we set out to re-evaluate this individual's REMS status 30 years after the original report, and formally assess his cognitive abilities. METHODS: Four whole-night polysomnographic sleep recordings were conducted to evaluate sleep architecture. Sleep scoring was performed according to the American Academy of Sleep Medicine (AASM) guidelines. Cranial Computed Tomography (CT) imaging was performed, as well as formal neuropsychological testing to evaluate cognitive functions. RESULTS: Y.C. averaged 4.5% of sleep time in REMS, corresponding to the 0.055 percentile of normal values for his age. Furthermore, residual REMS episodes were short and only occurred towards the end of the night. CT imaging revealed damage and metallic fragments in pons, cerebellum, and thalamus. Neuropsychological evaluation demonstrated average to high-average cognitive skills, normal memory, and motor difficulties including speech and left hand dyspraxia. CONCLUSIONS: To our knowledge, this is the only case where REMS loss resulting from pontine lesion was re-evaluated after many years. We find a near-total absence of REMS with no signs of significant compensation throughout adult life, along with normal cognitive status. The results provide a unique perspective on the ongoing debate regarding the functional role of REMS in supporting cognition.

Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes.

Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (Gϕ) which spontaneously develop in vitro without additional growth factors or cytokines. Gϕ are CD66b(+)/CD63(+)/MPO(+)/LC3B(+) and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS) production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕ formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished Gϕ formation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24 h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phox resembled Gϕ formed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕ formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b(+)/LC3B(+) Gϕ and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕ development.

Clinical implications of sleep disordered breathing in acute myocardial infarction.

BACKGROUND: Sleep disordered breathing (SDB), characterized by nightly intermittent hypoxia, is associated with multiple pathophysiologic alterations that may adversely affect patients with acute myocardial infarction (AMI). This prospective study investigated whether the metabolic perturbations associated with SDB are present when these patients develop AMI and if they affect clinical outcomes. METHODS: We prospectively enrolled 180 AMI patients. SDB was defined as oxygen desaturation index (ODI) >5 events/hour based on a Watch Pat-100 sleep study. Blood samples were obtained for high-sensitivity C-reactive protein (hs-CRP) and markers of oxidative stress (lipid peroxides [PD] and serum paraoxonase-1 [PON-1] (arylesterase activity). Echocardiography was performed to evaluate cardiac dimensions and pulmonary artery systolic pressure. RESULTS: SDB was present in 116 (64%) patients. Hs-CRP levels, PD and PON-1 were similar in patients with and without SDB. Echocardiography revealed higher left atrial dimension (4.1 ± 0.5 vs 3.8 ± 0.5 cm; P = 0.003) and a significant positive correlation between ODI and pulmonary artery systolic pressure (r = 0.41, P<0.0001). After a median follow up of 68 months, no significant differences were observed between the study groups with regard to clinical outcomes, including death, heart failure, myocardial infarction and unstable angina. CONCLUSION: There is a high prevalence of previously undiagnosed SDB among patients with AMI. SDB in the setting of AMI is associated with higher pulmonary artery systolic pressure. SDB was not associated with adverse clinical outcomes.

Endothelial progenitor cells in acute myocardial infarction and sleep-disordered breathing.

RATIONALE: Mobilization and functions of endothelial progenitor cells (EPCs) are increased in patients with acute myocardial infarction (AMI). Yet, sleep-disordered breathing (SDB) is highly prevalent in patients with AMI. OBJECTIVES: To compare EPC numbers and functions in patients with AMI with SDB (AMI-SDB) and without SDB (AMI-only) and to determine the effects of intermittent hypoxia (IH) in vitro on EPC proliferative and angiogenic properties. METHODS: Forty male patients with AMI underwent a whole-night sleep study using ambulatory monitoring. Nineteen had SDB (oxygen desaturation index > 5 events/h). AMI-SDB and AMI-only patients were matched by age, body mass index, blood chemistry, and comorbidities. Blood samples were analyzed by flow cytometry, endothelial cell colony-forming units (EC-CFU), paracrine measures, blood chemistry, and oxidative stress, inflammatory, and angiogenic markers. Effects of IH in vitro were studied in 12 healthy subjects. MEASUREMENTS AND MAIN RESULTS: Circulating EPCs (CD34(+)/KDR(+)), angiogenic T cells (CD3(+)/CD31(+)/CXCR4(+)), and vascular endothelial growth factor in monocytes were significantly higher in AMI-SDB patients, whereas plasma stromal cell-derived factor (SDF)-1α levels were significantly lower. Also, EC-CFU numbers and EC-CFU paracrine effects on endothelial tube formation were significantly higher in AMI-SDB compared with AMI-only patients. Similarly, in cell cultures from healthy subjects, EC-CFU numbers and their paracrine effects on endothelial tube formation were increased after exposure to IH in vitro compared with normoxia. CONCLUSIONS: Coexistent mild to moderate SDB in patients with AMI increased the mobilization, proliferative and angiogenic capacities of EPCs, angiogenic T-cell numbers, and vascular endothelial growth factor expression in monocytes compared with patients with AMI without SDB. IH in vitro had similar effects on healthy EPC functions.

Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling.

BACKGROUND: Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA) characterized by nightly intermittent hypoxia (IH). This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated. METHODS: Purified neutrophils were exposed to IH and compared to normoxia and to sustained hypoxia (SH) using a BioSpherix-OxyCycler C42 system. Bax and Mcl-1 levels, and p38MAPK and ERK1/2 phosphorylation were determined by western blotting. Also, Bax/Mcl-1 expression and Bax translocation to the mitochondria were assessed by confocal microscopy in pre-apoptotic neutrophils, before the appearance of apoptotic morphology. Co-localization of Bax and mitochondria was quantified by LSM 510 CarlZeiss MicroImaging using Manders Overlap Coefficient. A paired two-tailed t test, with Bonferroni correction for multiple comparisons, was used for statistical analysis. RESULTS: Compared to normoxia, IH and SH up-regulated the anti-apoptotic Mcl-1 by about 2-fold, down-regulated the pro-apoptotic Bax by 41% and 27%, respectively, and inhibited Bax co-localization with mitochondria before visible morphological signs of apoptosis were noted. IH induced ERK1/2 and p38MAPKs phosphorylation, whereas SH induced only p38MAPK phosphorylation. Accordingly, both ERK and p38MAPK inhibitors attenuated the IH-induced Mcl-1 increase. In SH, only p38MAPK inhibition decreased Mcl-1 expression. Similar to neutrophils of healthy subjects exposed to IH (0.97± 0.2), in OSA neutrophils, Bax/Mcl-1 ratio was significantly lower compared to normoxic controls (1.0±0.5 vs.1.99±0.3, p=0.015), and Bax did not co-localize with mitochondria. CONCLUSIONS: These findings suggest that decreased Bax/Mcl-1 balance promotes neutrophil survival in IH in-vitro as well as in OSA patients. Moreover, Bax/Mcl-1 protein function in IH and SH might be regulated by different signal transduction pathways, highlighting a novel regulatory function through ERK1/2 signaling in IH.

Screening for sleep disordered breathing among applicants for a professional driver's license.

OBJECTIVES: Individuals with sleep disordered breathing (SDB) are at high risk for falling asleep while driving. The aim of this study was to identify variables that would predict SDB among healthy young applicants for a professional driver's license. METHOD: A total of 301 applicants for a driver's license completed self-administered questionnaires. Sleep was recorded for one night with the Watch peripheral arterial tone-100. To identify possible predictors of SDB (Respiratory Disturbance Index >15), we employed new statistical methods. RESULTS: The following items were identified as significant predictors of SDB: body mass index, age, Mini Sleep Questionnaire, smoking, father snoring, afternoon nap-taking, and falling asleep while traveling as a passenger. Moderate or severe SDB was prevalent in at least 25% of the applicants. CONCLUSION: New statistical methods revealed that a combination of questions related to sleep habits, complaints, and demographic data predicted most of the clinically significant SDB.

Obesity plays an independent worsening modifying effect on nocturnal hypoxia in obstructive sleep apnea.

OBJECTIVE: Obesity's association with obstructive sleep apnea syndrome (OSAS) is well established. The aim of this study was to examine the impact of obesity on nocturnal hypoxia. METHODS: Twenty two thousand seven hundred and ninety three patients aged 21-80 years who underwent whole night polysomnography during 2000-2009 were included in the study. For each patient, percent sleep time spent with oxyhemoglobin desaturation lower than 90% was calculated (%NODP). Nocturnal hypoxia was analyzed by gender, age, OSAS severity, and body mass index (BMI) categories. RESULTS: Nocturnal hypoxia was positively correlated with BMI, OSAS severity, and age, but not with gender. The differences in %NODP between the two obese groups and non-obese varied from 0.55% to more than a 20% increase and were correlated with age and OSAS severity. The %NODP ratio between the two obese groups and the non-obese was independent of age and OSAS severity. There was a nearly twofold increase in %NODP in obese patients and a threefold increase in the morbidly obese relative to the non-obese. CONCLUSIONS: Our results indicate that obesity plays an independent worsening modifying effect on nocturnal hypoxia in OSAS.

Age- and gender-related characteristics of obstructive sleep apnea.

PURPOSE: The present study attempted to characterize the phenotype of men and women of different ages with a laboratory diagnosis of obstructive sleep apnea (OSA) using demographic, subjective complaints and medical history and to determine the best fitting apnea-hypopnea index (AHI) cutoff point for OSA diagnosis in each group. METHODS: Data collected from 23,806 patients examined by a whole-night polysomnography were retrospectively analyzed. First, descriptive analysis was used to determine the gender-specific relationship between AHI and age, then binary logistic regression was used to determine the best fitting gender- and age-specific AHIs and the predictors of OSA in each age and gender group. RESULTS: Of the total number of patients, 70.7% had AHI >10, and men had consistently higher AHI than women. OSA severity rose linearly with age in normal-weight and obese women and in normal-weight men. The best fitting AHI cutoff point increased with age in both genders. Obesity and snoring were significant predictors of OSA in all age by gender groups, while hypertension and excessive daytime sleepiness were common to all men and the two older women groups. Insomnia-related complaints were negative predictors of obstructive sleep apnea syndrome (OSAS) in some of the groups. CONCLUSIONS: OSAS severity varies with age in both genders, while women have less severe syndrome in all ages. Obesity, snoring, hypertension, and excessive daytime sleepiness are OSAS predictors in both genders, while insomnia-related complaints are negative predictors.

Sleep profile and symptoms of sleep disorders in patients with stable mild to moderate chronic obstructive pulmonary disease.

BACKGROUND: Sleep problems associated with chronic obstructive pulmonary disease may have an important impact on quality of life and health outcome measures in patients. The aim of this study was to prospectively assess differences in symptom profile and polysomnographic parameters in patients with stable mild to moderate COPD and age, gender, and body-mass-index matched controls without airflow obstruction. METHODS: The Sleep Disorders Questionnaire was administered to both patients and controls prior to clinical and polysomnographic evaluation. Responses obtained from the questionnaire were used to construct four independent symptom scales: sleep apnea, periodic limb movement syndrome, psychiatric sleep disorder, and narcolepsy. Associations between each diagnostic scale and sleep parameters were considered by means of multiple analyses of covariance. RESULTS: Fifty-two patients with mild-to-moderate COPD (age 62±8 years, BMI 29±7 kg/sqm) and 52 age, gender, and body-weight matched controls without COPD were studied. Patients with COPD had overall lower sleep efficiency, a lower total sleep time, and lower mean overnight oxygen saturation compared to controls. Patients with COPD were significantly more likely to report symptoms such as insomnia and difficulty in initiating and maintaining sleep, resulting in overall higher psychiatric sleep disorder scale scores in patients compared with controls. Minimum oxygen saturation was an independent predictor for all symptom scales. After correcting for potentially confounding factors, including pack/years of smoking, total sleep time, sleep efficiency, arousal index, mean and minimum oxygen saturation, and apnea-hypopnea-index, the between group-differences for both the periodic limb movement and psychiatric sleep disorder scale scores remained statistically significant. CONCLUSIONS: We observed significant differences in both quantity and quality of sleep between patients with stable mild to moderate chronic obstructive pulmonary disease and respective controls.

Molecular pathways of spontaneous and TNF-{alpha}-mediated neutrophil apoptosis under intermittent hypoxia.

Apoptosis of polymorphonuclear cells (PMNs) is a fundamental mechanism to halt inflammation. It limits the lifespan of PMNs and thereby decreases tissue injury. In PMNs, unlike in other cells, hypoxia profoundly inhibits apoptosis. However, most studies investigating hypoxic effects on the functioning of PMN focus on acute or chronic sustained hypoxia. Thus, the mechanisms by which intermittent hypoxia (IH) affects PMN apoptosis are not known. Flow cytometry and Western blotting were used to evaluate mechanisms of constitutive and TNF-α-mediated PMN apoptosis in IH. The levels of NF-κB, p38 mitogen-activated protein kinase (MAPK), TNF receptor-2 (TNFR-2), intracellular IL-8 and its surface receptor CXCR2, were determined. Specific NF-κB (gliotoxin and parthenolide) and p38MAPK (SB202190) inhibitors were also used. TNF-α-mediated PMN apoptosis was concentration-dependent; low concentration increased PMN survival, whereas higher concentrations accelerated apoptosis. However, at all TNF-α concentrations, PMN survival was higher after four IH cycles than in normoxia. However, increasing the IH cycles to six abolished the pro-apoptotic/anti-apoptotic effects of TNF-α. Also, IH increased TNRF2 expression, nuclear NF-κB translocation, p38MAPK phosphorylation, and expression of IL-8 and CXCR2. The NF-κB inhibitors gliotoxin and parthenolide increased apoptosis and decreased IL-8 and CXCR2 expression. Also, the p38MAPK inhibitor SB202190 increased apoptosis and decreased IL-8 expression but had no effect on CXCR2 expression. Collectively, these findings provide insights into the mechanisms that prolong PMN survival after IH exposure and demonstrate the essential role played by NF-κB, the p38MAPK signaling pathway, and downstream genes in this process.